Nitric oxide synthase Information & Nitric oxide synthase Links at HealthHaven.com

Nitric oxide synthases (EC 1.14.13.39) (NOSs) are a family of eukaryotic enzymes that catalyze the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule, having a vital role in many biological processes.

[edit] Purposes

NOS is an enzyme in the body that contributes to transmission from one neuron to another, to the immune system and to dilating blood vessels. It does so by synthesis of nitric oxide (NO) from the terminal nitrogen atom of arginine in the presence of NADPH and dioxygen (O₂). NOS is the only known enzyme that binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH₄) and calmodulin.

NO activates Guanylate cyclase, which induces smooth muscle relaxation by:

Increased intracellular cGMP, which inhibits calcium entry into the cell, and decreases intracellular calcium concentrations
Activation of K+ channels, which leads to hyperpolarization and relaxation
Stimulates a cGMP-dependent protein kinase that activates myosin light chain phosphatase, the enzyme that dephosphorylates myosin light chains, which leads to smooth muscle relaxation.

[edit] Classification

Different members of the NOS family are encoded by separate genes. ^[1]. NOS is one of the most regulated enzymes in biology. There are three known isoforms, two are constitutive (cNOS) and the third is inducible (iNOS). ^[2]. Cloning of NOS enzymes indicates that, cNOS include both brain constitutive (NOS1) and endothelial constitutive (NOS3), the third is the inducible (NOS2) gene. ^[3]. Recently, NOS activity has been demonstrated in several bacterial species, including such notorious pathogens as Bacillus anthraces and Staphylococcus aureus. ^[4]^[5]. Bacterial NOS (bNOS) has been shown to protect bacteria against oxidative stress, diverse antibiotics, and host immune response. ^[6]^[7]. The different forms of NO synthase have been classified as follows:

Name	Gene(s)	Location	Function
Neuronal NOS (nNOS or NOS1)	NOS1	nervous tissue skeletal muscle type II	cell communication
Inducible NOS (iNOS or NOS2)	NOS2A, NOS2B, NOS2C	immune system cardiovascular system	immune defence against pathogens
Endothelial NOS (eNOS or NOS3 or cNOS)	NOS3	endothelium	vasodilation
Bacterial NOS (bNOS)	multiple	various Gram(+) species	defense against oxidative stress, antibiotics, immune attack

[edit] nNOS

Neuronal NOS (nNOS) produces NO in nervous tissue in both the central and peripheral nervous system. Neuronal NOS also performs a role in cell communication and is associated with plasma membranes. nNOS action can be inhibited by NPA (N-propyl-L-arginine). This form of the enzyme is specifically inhibited by 7-nitroindazole.^[8]

[edit] iNOS

As opposed to the critical calcium-dependent regulation of constitutive NOS enzymes (nNOS and eNOS), iNOS has been described as calcium-insensitive, likely due to its tight non-covalent interaction with calmodulin (CaM) and Ca²⁺. While evidence for ‘baseline’ iNOS expression has been elusive, IRF-1 and NF-κB-dependent activation of the inducible NOS promoter supports an inflammation mediated stimulation of this transcript.

From a functional perspective, it is important to recognize that induction of the high-output iNOS usually occurs in an oxidative environment, and thus high levels of NO have the opportunity to react with superoxide leading to peroxynitrite formation and cell toxicity.

These properties may define the roles of iNOS in host immunity, enabling its participation in anti-microbial and anti-tumor activities as part of the oxidative burst of macrophages.^[9]

[edit] eNOS

Main article: Endothelial NOS

Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in blood vessels and is involved with regulating vascular function. A constitutive Ca²⁺ dependent NOS provides a basal release of NO. eNOS is associated with plasma membranes surrounding cells and the membranes of Golgi bodies within cells.

[edit] Chemical reaction

Nitric oxide synthase produces NO by catalysing a five-electron oxidation of a guanidino nitrogen of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two successive monooxygenation reactions producing N^ω-hydroxy-L-arginine (NOHLA) as an intermediate. 2 mol of O₂ and 1.5 mol of NADPH are consumed per mole of NO formed.

L-Arg + NADPH + H⁺ + O₂ → NOHLA + NADP⁺ + H₂O

NOHLA + ½ NADPH + ½ H⁺ + O₂ → L-citrulline + ½ NADP⁺ + NO + H₂O

[edit] Structure

NOSs can be dimeric, calmodulin-dependent or calmodulin-containing cytochrome p450-like hemoprotein that combines reductase and oxygenase catalytic domains in one dimer, bear both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), and carry out a 5`-electron oxidation of non-aromatic amino acid arginine with the aid of tetrahydrobiopterin. ^[10].

All three isoforms (each of which is presumed to function as a homodimer during activation) share a carboxyl-terminal reductase domain homologous to the cytochrome P450 reductase. They also share an amino-terminal oxygenase domain containing a heme prosthetic group, which is linked in the middle of the protein to a calmodulin-binding domain. Binding of calmodulin appears to act as a "molecular switch" to enable electron flow from flavin prosthetic groups in the reductase domain to heme. This facilitates the conversion of O₂ and L-arginine to NO and L-citrulline. The oxygenase domain of each NOS isoform also contains an BH₄ prosthetic group, which is required for the efficient generation of NO. Unlike other enzymes where BH₄ is used as a source of reducing equivalents and is recycled by dihydrobiopterin reductase (EC 1.5.1.33), BH₄ activates heme-bound O₂ by donating a single electron, which is then recaptured to enable nitric oxide release.

The first nitric oxide synthase to be identified was found in neuronal tissue (NOS1 or nNOS); the endothelial NOS (eNOS or NOS3) was the third to be identified. They were originally classified as "constitutively expressed" and "Ca²⁺ sensitive" but it is now known that they are present in many different cell types and that expression is regulated under specific physiological conditions.

In NOS1 and NOS3, physiological concentrations of Ca²⁺ in cells regulate the binding of calmodulin to the "latch domains", thereby initiating electron transfer from the flavins to the heme moieties. In contrast, calmodulin remains tightly bound to the inducible and Ca²⁺-insensitive isoform (iNOS or NOS2) even at a low intracellular Ca²⁺ activity, acting essentially as a subunit of this isoform.

Nitric oxide may itself regulate NOS expression and activity. Specifically, NO has been shown to play an important negative feedback regulatory role on NOS3, and therefore vascular endothelial cell function. This process, known formally as S-nitrosation (and referred to by many in the field as S-nitrosylation), has been shown to reversibly inhibit NOS3 activity in vascular endothelial cells. This process may be important because it is regulated by cellular redox conditions and may thereby provide a mechanism for the association between "oxidative stress" and endothelial dysfunction. In addition to NOS3, both NOS1 and NOS2 have been found to be S-nitrosated, but the evidence for dynamic regulation of those NOS isoforms by this process is less complete. In addition, both NOS1 and NOS2 have been shown to form ferrous-nitrosyl complexes in their heme prosthetic groups that may act partially to self-inactivate these enzymes under certain conditions. The rate-limiting step for the production of nitric oxide may well be the availability of L-arginine in some cell types. This may be particularly important after the induction of NOS2.

[edit] References

^ Tylor B.S., Kion Y.M., Wang Q.I., Sharpio R.A., Billiar T.R. and Geller D.A. (1997)Nitric oxide down regulates hepatocyte-inducible nitric oxide synthase gene expression. Arch. Surg. 1, (32). Nov.; 1177-1182
^ Majano P., Lara-Pezzi E., Lopez-Cabrera M., Apolinario A., Moreno-Otero R. and Garcia-Monzon C. (2001): Hepatitis B virus X protein transactivates inducible nitric oxide synthase gene promoter through the proximal nuclear factor kappa B-binding site: evidence that cytoplasmic location of X protein is essential for gene transactivation. Hepatology Dec; 34(6): 1218-1224.)
^ Gilkeson G., Mudgetts J., Seldin M.F., Ruiz P., Alexander A., Misukonis M.A., Pistesky D.S. and Weinberg J.B. (1997): Clinical and serologic manifestation of autoimmune disease in MRL-Mice lacking Nitric Oxide Synthase Type 2. J. Exp. Med. 186 (3): August; 4: 365-373.
^ Gusarov I, Starodubtseva M, Wang ZQ, McQuade L, Lippard SJ, Stuehr DJ, and Nudler E. Bacterial nitric-oxide synthases operate without a dedicated redox partner. J Biol Chem. 2008 May 9;283(19):13140-7
^ Shatalin K, Gusarov I, Avetissova E, Shatalina Y, McQuade LE, Lippard SJ, and Nudler E. Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages. Proc Natl Acad Sci U S A. 2008 Jan 22;105(3):1009-13
^ Gusarov I, Nudler E. NO-mediated cytoprotection: instant adaptation to oxidative stress in bacteria. Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13855-60
^ Gusarov I, Shatalin K, Starodubtseva M, and Nudler E. Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics. Science. 2009 Sep 11;325(5946):1380-4
^ Southan GJ, Szabó C (February 1996). "Selective pharmacological inhibition of distinct nitric oxide synthase isoforms". Biochem. Pharmacol. 51 (4): 383–94. doi:10.1016/0006-2952(95)02099-3. PMID 8619882.
^ Mungrue, et al., Heart Fail Rev. 2002 Oct;7(4):407-22.
^ Chinje E.C. and Stratford I.J. (1997): Role of nitric oxide in growth of solid tumors: a balancing act. Essays Biochem.; 32: 61-72.

[edit] External links

IPR004030 - InterPro entry for NOS
PDB 1DWX - X-ray structure of murine iNOS oxygenase domain in complex with NOHLA and BH₄
PDB 1F20 - X-ray structure of rat nNOS reductase FAD/NADP⁺ domain
The Nobel Prize in Physiology or Medicine 1998
University of Edinburgh, School of Chemistry - NO Synthase
MeSH Nitric+oxide+synthase
Nitric Oxide Synthase in Proteopedia

[0] Tylor B.S., Kion Y.M., Wang Q.I., Sharpio R.A., Billiar T.R. and Geller D.A. (1997)Nitric oxide down regulates hepatocyte-inducible nitric oxide synthase gene expression. Arch. Surg. 1, (32). Nov.; 1177-1182

[1] Majano P., Lara-Pezzi E., Lopez-Cabrera M., Apolinario A., Moreno-Otero R. and Garcia-Monzon C. (2001): Hepatitis B virus X protein transactivates inducible nitric oxide synthase gene promoter through the proximal nuclear factor kappa B-binding site: evidence that cytoplasmic location of X protein is essential for gene transactivation. Hepatology Dec; 34(6): 1218-1224.)

[2] Gilkeson G., Mudgetts J., Seldin M.F., Ruiz P., Alexander A., Misukonis M.A., Pistesky D.S. and Weinberg J.B. (1997): Clinical and serologic manifestation of autoimmune disease in MRL-Mice lacking Nitric Oxide Synthase Type 2. J. Exp. Med. 186 (3): August; 4: 365-373.

[3] Gusarov I, Starodubtseva M, Wang ZQ, McQuade L, Lippard SJ, Stuehr DJ, and Nudler E. Bacterial nitric-oxide synthases operate without a dedicated redox partner. J Biol Chem. 2008 May 9;283(19):13140-7

[4] Shatalin K, Gusarov I, Avetissova E, Shatalina Y, McQuade LE, Lippard SJ, and Nudler E. Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages. Proc Natl Acad Sci U S A. 2008 Jan 22;105(3):1009-13

[5] Gusarov I, Nudler E. NO-mediated cytoprotection: instant adaptation to oxidative stress in bacteria. Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13855-60

[6] Gusarov I, Shatalin K, Starodubtseva M, and Nudler E. Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics. Science. 2009 Sep 11;325(5946):1380-4

[Southan-7] Southan GJ, Szabó C (February 1996). "Selective pharmacological inhibition of distinct nitric oxide synthase isoforms". Biochem. Pharmacol. 51 (4): 383–94. doi:10.1016/0006-2952(95)02099-3. PMID 8619882.

[8] Mungrue, et al., Heart Fail Rev. 2002 Oct;7(4):407-22.

[9] Chinje E.C. and Stratford I.J. (1997): Role of nitric oxide in growth of solid tumors: a balancing act. Essays Biochem.; 32: 61-72.

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